EL MÉTODO COMPARATIVO
V. Contexto y diseño institucional
2. Requisitos metódicos del diseño institucional
Summary of Findings
This project examined the potential role of the extracellular matrix protein,
Slit2 and its receptor Roundabout (Robo) in reducing complications following
neurosurgical injury. We specifically examined the contribution of Slit2 in
reducing neuroinflammation and blood brain barrier (BBB) disruption after
surgical brain injury (SBI) using a rat model. The major observations and
conclusions obtained from the project have been summarized in this section.
Endogenous Slit2 has a Protective Function After SBI
Our findings from Chapter 3 show that endogenous Slit2 has a protective
function after SBI. We observed that endogenous Slit2 was upregulated after SBI
starting at 24 h upto 7 days after the injury, and knockdown of endogenous Slit2
using siRNA worsened brain edema and neurological function after SBI. In
addition, we observed that Slit2 was expressed by neurons and astrocytes after
the injury, which we speculate is the source for increase in endogenous levels of
Slit2 as a protective response to injury. These findings complement previous
study in which Slit2 was shown to be increased after traumatic brain injury
(Hagino et al., 2003). Although the function of endogenous Slit2 in adults after
brain injury has not been explored, our findings that endogenous Slit2
knockdown worsened outcomes after SBI suggests a protective role of Slit2
Recombinant Slit2 Reduces SBI Induced Complications
Our findings from Chapter 3 show that recombinant Slit2 pretreatment
reduced SBI induced brain edema and neurological deficits in SBI rats. The
improved outcomes with recombinant Slit2 pretreatment was associated with
reduced neuroinflammation and peripheral leukocyte migration to the perisurgical
site after SBI. Post-operative brain edema is one of the major complications
encountered in neurosurgical patients (Wong et al., 2012). Neuroinflammation
and disruption of the BBB can worsen brain edema and post-operative
neurological function (Bruder and Ravussin, 1999, Hyong et al., 2008, Huang et
al., 2015), which were both attenuated with recombinant Slit2. Additionally, our
results from Chapter 4 show that recombinant Slit2 reduced BBB permeability
and stabilized the BBB tight junction at the perisurgical site after SBI. These
findings support the therapeutic potential of recombinant Slit2 to reduce
neuroinflammation and BBB disruption following brain injury.
Robo Receptor Mediated Signaling Pathways Contribute to the Protective Effects of Slit2
The Robo family of receptors is known to be activated by the Slit2 ligand
(Guan and Rao, 2003, Ballard and Hinck, 2012). We evaluated the role of Robo1
and Robo4 receptors in Slit2 mediated protection after SBI. Our findings in
Chapter 3 show that Robo1 was expressed by peripheral immune cells that
infiltrated at the perisurgical site after SBI. Our results suggest that inactivation of
Cdc42 via Robo1-srGPA1 pathway contributed to the inhibition of peripheral
reversed the anti-migratory effect of recombinant Slit2 on the peripheral immune
cells. Likewise, our findings in Chapter 4 show that Robo4 was expressed by the
endothelial cells. The BBB protective function of recombinant Slit2 was
associated with Robo4-paxillin mediated Rac1 activation, and knockdown of
Robo4 or paxillin reversed Slit2 mediated Rac1 activation. These findings
suggest that Robo1 and Robo4 receptors are primarily involved in mediating the
protective effects of Slit2 by modulating the downstream signaling pathways.
Scientific Contribution
This project was focused on studying SBI, which is an important clinical
problem that has largely been overlooked due to a lack of understanding of the
occurrence of the problem. Even though adverse events such as brain edema
and post-operative neurological deficits often occur following neurosurgical
procedure (Bruder and Ravussin, 1999, Rolston et al., 2014), the
pathophysiology of SBI is often not studied. This project explored two major
pathophysiological events that occur after SBI. First, the role of peripheral
immune cell migration in mediating neuroinflammation after SBI was examined
particularly with a focus on Robo1 mediated immune cell migration to the injury
site. Second, post-operative brain edema resulting from BBB junction disruption
was examined with a focus on restoring BBB stability after SBI.
This project has identified novel molecular mechanisms involved in Slit2
mediated neuroprotection. Slit2 has primarily been studied in the developing
nervous system and few studies have explored the role of Slit2 in adult brain
shown that Slit2 reduced injury outside the CNS in animal models of renal and
lung inflammation (Kanellis et al., 2004, Ye et al., 2010), the function of Slit2 after
brain injury has been relatively unexplored. Slit2 was shown to be increased after
experimental traumatic brain injury (Hagino et al., 2003) and exogenous Slit2
was beneficial in a cerebral ischemia rodent model (Altay et al., 2007). However,
functional significance of Slit2 after brain injury and the protective mechanism of
Slit2 were not determined. Our findings in this project provide evidence that Slit2
has a protective role after brain injury. We have elucidated previously unknown
dual function of Slit2 after brain injury, which includes reducing
neuroinflammation by inhibiting the migration of peripheral immune cells and anti-
permeability effects by stabilizing the BBB tight junction. Furthermore, our
findings demonstrate that the protective mechanism of Slit2 is mediated via
Robo1 receptor expressed by peripheral immune cells and Robo4 receptor
expressed by BBB endothelial cells. These novel findings show that exogenous
Slit2 administration targets two major pathophysiological events after SBI and thereby augments the body’s endogenous protective response against SBI.
Overall, these findings provide evidence that Slit2 has beneficial role after
SBI and is a potentially suitable therapeutic candidate to reduce deleterious
consequences after SBI and possibly other brain injuries with similar pathologies.
Limitations and Future Directions
Role of Other Members of the Slit Protein Remains to be Elucidated
This project primarily focused on exploring the role of Slit2 after SBI even
though there are three known isoforms of Slit proteins (Slit1-Slit3) (Guan and
Rao, 2003, Hohenester, 2008). Studies show that other Slit members may
regulate processes in the CNS too. For instance, Slit1 is predominantly
expressed in the developing CNS and Slit3 has been reported to have anti-
permeability effects following VEGF induced retinal hyperpermeability (Marillat et
al., 2002, Jones et al., 2008). Given these findings, it is likely that Slit1 and Slit3
may be involved during recovery after CNS injury. However, Hagino et al.
observed that Slit2 was predominantly expressed surrounding the lesion after
traumatic brain injury in mice while the expression of Slit1 and Slit3 was much
weaker (Hagino et al., 2003). In accordance, we observed that the expression of
Slit2 was increased after SBI. However, we did not evaluate if Slit1 and Slit3
exhibit any changes after SBI. Neither did we explore therapeutic potential of the
other Slit members after SBI. A detailed examination of all members of the Slit
family after brain injury is warranted.
Alternate Pathways in Slit2 Mediated Protection Need to be Evaluated
This project examined two primary pathways in Slit2 mediated protection
after SBI which included the Robo1-srGAP1 pathway regulating
neuroinflammation and the Robo4-paxillin pathway maintaining the endothelial
pathways, which was not explored in this project.
For instance, (1) Downstream effectors such as Abelson kinase (Abl) and
Enabled (Ena) can interact with the intracellular motif of Robo and regulate cell
migration. Abl antagonizes the repulsive effects of Robo whereas, Ena enhances
Robo mediated repulsive signaling pathway (Ballard and Hinck, 2012). (2) We
measured Cdc42 activity as the downstream substrate of srGAP1 but other small
GTPases including RhoA and Rac1 that regulate cellular actin organization and
motility were not evaluated in this project (Wojciak-Stothard and Ridley, 2002,
Waschke et al., 2004). (3) Previous studies have shown that Slit2 can inhibit
chemokine induced Src kinase activity and Lck kinase activity which have been
reported to regulate cell migration (Prasad et al., 2007). (4) The endothelial
barrier protective effect of Slit2 may be mediated by modulation of
matrixmetalloproteinase (MMP)-9 activity which was not explored (Cai et al.,
2015). (5) Lastly, using knockout or transgenic animals would strengthen the
findings in our project. It was previously observed that Robo4 null mice had
increased severity of lung injury than Robo4+/+ mice when subjected to lung
inflammation (London et al., 2010), which proposes a protective function for Slit2-
Robo4. However, Slit2 transgenic mice were more susceptible to collagenase
induced ICH and had larger hemorrhagic volumes compared to control mice
(Han and Geng, 2011). The findings in this study conflict with the protective
effects of Slit2 observed in other animal models of CNS injury (Altay et al., 2007,
Sherchan et al., 2015). This suggests that endogenous Slit2 overexpression and
endothelial cells.
Additionally, the findings in these studies could be attributed to differences
in the animal model used, differential mechanism of the endogenous versus
exogenous Slit2, or due to differences in the magnitude of exposure to Slit2 in
transgenic animals as opposed to exogenous recombinant Slit2 administration.
Although the temporal pattern and duration of Slit2 expression in transgenic
animals subjected to injury and the mechanism of injury was not explored, these
findings necessitate further exploration of the function of Slit2 after brain injury.
Detailed Exploration of the Robo Receptor Subtypes is Required
The effect of Slit2 may depend on the type of receptor predominantly
expressed following an injury. Furthermore, the expression and function of Robo
receptors may be tissue specific and context dependent. For instance, Slit2
reduced permeability in human pulmonary microvascular endothelial cells
(PMECs) that predominantly express Robo4 (Gorbunova et al., 2013), but the
anti-permeability effect of Slit2 was not observed in the human umbilical vein
endothelial cells (HUVECs) which express similar levels of Robo4 and Robo1
receptors (Gorbunova et al., 2013). Therefore, an in depth understanding of the
expression of Robo receptors and changes that may occur after SBI is required.
Upstream Regulators of Slit2 Need to be Explored
The transcription factor such as Pax6 has been shown to regulate the
for instance hypermethylation of the gene in systemic tumors and glioma have
been observed (Dallol et al., 2003). Our findings showed that endogenous Slit2
levels increased after SBI as a protective response to counteract the
consequences of SBI. However, the upstream regulators for Slit2 were not
examined. Further exploration into the regulation of endogenous Slit2 is essential
to understanding this response.
Conclusion
Our findings suggest that Slit2 has a beneficial role against
neuropathological consequences after SBI. The protective effect of Slit2 against
SBI was possibly mediated by its anti-migratory function against peripheral
leukocytes and by endothelial barrier stabilization dependent on the Robo
receptors. The dual protective function of Slit2 makes it a potential therapeutic
option to reduce neurosurgical injury and improve post-operative outcomes in
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